PROJECT SUMMARY/ABSTRACT Inflammatory bowel diseases (IBDs) are characterized by chronic inflammation of the gastrointestinal tract. The microbes inhabiting the mammalian gastrointestinal tract, collectively known as the intestinal microbiota, likely play a role in the genesis and/or potentiation of this inflammation. These microbes also play an opposing role in the establishment of intestinal homeostasis and protection from IBD. Understanding the mechanisms of inter-kingdom communication that must occur between the host and its intestinal microbiota during disease pathogenesis is crucial for the development of therapeutics that can prevent microbe-driven disease. Previous study of this inter-kingdom communication has focused primarily on the role of Toll-like receptors and NOD-like receptors, germline encoded receptors expressed in eukaryotic cells that bind to conserved protein or nucleic acid motifs found in bacteria. Here, we show for the first time the presence of microbe-derived cyclic dinucleotides (CDNs) in the mammalian gastrointestinal tract. We show that in vitro, model commensal microorganisms package these CDNs into outer membrane vesicles (OMVs), which can fuse with host cells to deliver their contents. Microbe-derived CDNs have been shown to bind to the intracellular eukaryotic protein STING in the context of pathogenic bacterial infections. Ligation of STING by CDNs leads to interferon production, the activation of NF-?B pro-inflammatory and pro-survival signaling, and the activation of autophagy. Additionally, CDNs have been used experimentally as effective mucosal vaccine adjuvants, resulting in enhanced recruitment and activation of innate immune cells. We therefore hypothesize that cyclic dinucleotides generated by the intestinal microbiota and packaged into OMVs activate an innate immune response in the host and are necessary for the establishment of intestinal homeostasis. We propose to test this hypothesis with two discrete aims: (1) to determine the role of OMV packaged CDNs in the activation of host innate immunity, and (2) to determine the effect of microbe-derived CDNs in a model of colitis. In aim 1, we propose to examine the mechanism of host-microbe communication via CDN-containing OMVs in in vitro models of the intestinal epithelium and innate immune system. In aim 2, we propose to examine the role of CDNs in an in vivo model of colitis through use of a mouse model that is genetically modified to lack the CDN receptor. This work will open an exciting new area of research in the field of intestinal inflammation. Additionally, its completion will provide excellent technical and scientific training for the applicant, serving to develop her potential as an independent investigator.